JPS6035266A - Microcapsule for multi-kind antibody detection and inspection using the same - Google Patents

Abstract

PURPOSE:To make clear the agglutination pattern with a greater uniformity of the sensitivity by mixing a microcapsule uneven in the sensitivity level auxiliarily with a microcapsule singly sensitized with an antigen lacking the sensitivity level. CONSTITUTION:A microcapsule for multi-kind antibody detection as main one contains a microcapsule mixed and then, sensitized with more than one different kinds of antigens. Here, the different kinds of antigens are expressed by A and B and the main microcapsule shall reach the specified level in the sensitivity for the antigen component of A not for the antigen component of B. At this point, the main microcapsule mixed and then, sensitized with A and B is mixed with a microcapsule singly sensitized with the antigen component B as auxiliary microcapsule. As a result, the sensitivity of the antigen component B rises maintaining the initial sensitivity of the antigen component A thereby improving the uniformity of the sensitivity.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved microcapsule reagent capable of simultaneously detecting multiple antibodies, and a multiple antibody testing method using such a microcapsule reagent.

It is known that the reactivity between antigens such as pathogenic bacteria and viruses and antibodies generated by the antigens is extremely specific. In order to distinguish between serotypes by utilizing this extremely high serotype specificity, Japanese Patent Application Laid-Open No. 58-21565
Microcapsules sensitized with two or more antigens of different serotypes were used. According to this method, diagnosis is made based on the antigen-antibody reaction that strictly corresponds to the serotype.

Diagnostic accuracy is extremely high. The problem lies in how to equalize the sensitivity levels of different antigen components during sensitization. Having the same sensitivity level is an important condition for improving reproducibility, but since sensitization is performed with a foreign antigen, it is natural for each sensitivity level to generally vary. This is probably due to differences in the number and activity of bacteria depending on the growth rate and growth conditions of the bacteria themselves. In practice, only those sensitized microcapsules in which each of the antigen components has the same sensitivity level are selected and used, so microcapsules with variations in sensitization were treated as manufacturing losses.

The present inventors have found that even if microcapsules have uneven sensitivity levels, if they are supplemented with microcapsules sensitized with an antigen that lacks sensitivity levels, the sensitivity becomes more uniform. We obtained the knowledge that the agglomeration/zo-turn becomes clearer.

The microcapsules for detecting multiple antibodies according to the present invention include, as main microcapsules, microcapsules mixed with two or more different antigens and then sensitized.

In a group of microcapsules in which the sensitivity level to each antibody of the foreign antigen is uneven, the main microcapsule is mixed with an antigen whose sensitivity level is insufficient as at least one microcassette all auxiliary microcapsules sensitized alone. It is characterized by

For example, if the antigen component A has a desired sensitivity level and the sensitivity level of the antigen component B has not reached the former level, A and Bf: After mixing, the antigen component B The microcapsules sensitized alone are mixed together as auxiliary microcapsules. In this mixed microcapsule, antigen component B
An increase in sensitivity was achieved, but on the other hand, from the perspective of antigen component A, this was the same as an increase in noise, and it was thought that the sensitivity of antigen component A would decrease. However, surprisingly, no desensitization of antigen component A occurred and the initial sensitivity was substantially maintained. Contrary to the above, such an effect occurs even when antigen component A is insufficient at the sensitivity level.

It can be similarly obtained by mixing microcapsules sensitized with component A alone as auxiliary microcapsules. In addition, if only the A component of the main microcapsule has the desired sensitivity level, but the B and C components are insufficient, a product sensitized with the B component alone or a product sensitized with the C component alone may be used. It may be mixed as auxiliary microcapsules.

The auxiliary microcapsule is 5 times larger than the main microcapsule.
It is desirable to mix in a range of 0 volume or less, preferably 15 to 45 volume. When the amount of auxiliary microcapsules exceeds the upper limit, sensitivity decreases.

If a small amount is used, the defects of the main microcapsules themselves will be exposed.

Sensitizing antigens that can be used in the present invention are selected from a wide range of antigens, including hormones, drug metabolites, 2% foreign proteins, viruses, bacteria, cells, and antigens of human origin. Specifically, for example.

Treponema pallidum antigen, HB, antigen, Toxoplasma antigen. Mycoplasma antigen, Shigella, Leptospira,
Vibrio cholerae 1st class. Among these, the reagent of the present invention using Leptospira bacteria, which is an antigen component of a different serotype, as a sensitizing antigen has a particularly high titer. iI/i of these sensitizing antigens,
Depending on various conditions such as the type and the accuracy of the intended measurement,
It will be selected as appropriate.

Generally, for the solid content of microcassice cells.

It is within the range of 0.01 to 10% by weight.

The method for sensitizing microcapsules with antigens is described in Japanese Patent Application Laid-open No. 1983-
21565 in detail.

The microcapsules used as carriers in the present invention consist of a core of an oily substance and a wall material surrounding the core. The general M method is explained in detail in, for example, "Microcapsule" by Asashi Kondo, published by Nikkan Kogyo Shimbun (1972). Furthermore, detailed descriptions of specific oily substances, wall materials, various additives, etc. can be found in JP-A-57-196621 and JP-A-57-19662.

Well-known methods are used to sensitize microcapsules with antigens or antibodies, and methods using crosslinking agents are particularly convenient (see "Immobilized Enzyme" by Kazuchi Chibata, Kodansha (1975), etc.).

The microcapsules used as carriers are 0.85-1
Those having a specific gravity in the range of 25 and an average particle size in the range of about 0.5 to 20 μm, preferably 1 to 10 μm are suitable.

It is desirable that the solid content of the microcapsule carrier is usually within a range of about 1 to 3 by weight.

The reagent of the present invention is used in immunoassays in which antibodies are detected by observing aggregation images by applying the microticou method. According to the present invention, it is possible to detect multiple types of antibodies with a single reagent, and furthermore, when observing the immunological cross-reactivity characteristic of the reagent of the present invention, it is possible to detect bacteria with multiple serotype specificities. Infectious diseases can be accurately diagnosed in just one operation. In the present invention, by using a mixed system of main/auxiliary microcapsules, it is possible to increase production yields, which is very useful industrially.

The present invention will be explained in more detail with reference to Examples below.

Example Preparation of seed strain sensitized microcapsule A.

118g of noisonolopyrnaphthalene and chlorinated paraffin (degree of chlorination: 50%) Yonoguracnu 150) 13.2
025 g of oil-soluble red dye oleosole red BB (manufactured by Sumitomo Chemical) was dissolved in a mixed oil (specific gravity of about 110) with g. The obtained oily substance liquid was treated with maleic anhydride-methyl vinyl ether copolymer (GANTREZ AN-149
, General Aniline and Film Co. M) 2.59f:
Added to 75ml of water dissolved in 1K solution. The mixture was stirred and emulsified, and the size of the oil droplets was measured using a Coulter Counter Model TA-1 so that the average size was about 5 μm. To this was added a solution of 2.5 g of urea, 0.25 g of resorcinol, and 0.3 g of ammonium chloride dissolved in 25 ml of water.

Further, 50 ml of water was added to dilute the mixture, and 7 ml of a 37% formaldehyde aqueous solution was added, followed by reaction at 60° C. for 2 hours to perform microencapsulation. Thereafter, an aqueous IN sodium hydroxide solution was added to adjust the pH to 0 to prepare microcapsules.

The thus-prepared microcapsules were centrifuged and washed with physiological saline to remove unreacted residues.

The microcapsule particles were dispersed in physiological saline so that the concentration thereof was 10 parts, and this was designated as microcapsule A.

Preparation of microcapsule reagent A sensitized with two bacterial strains: 1.59't of the obtained microcapsules were taken and dispersed in 10 ml of physiological saline.

Next, 100 μβ of a 25% glutaraldehyde aqueous solution was mixed, reacted at room temperature for 1 hour, and washed by centrifugation.

Redispersed in 10 ml of saline. Leptospira autumnalis strain A was grown in Kortov's medium (containing 10% normal rabbit serum) and centrifuged at ft9.0.0 Orpm for 20 minutes (5°C) on the 6th to 10th day of culture. The precipitated bacterial cells obtained were washed twice with physiological saline. Next, it was suspended in physiological saline and crushed for 10 minutes using a 20 kHz sonic crusher (manufactured by Otake Seisakusho).

Centrifugation supernatant (5,000 rpm 10 minutes) was measured at 1°C using a spectrophotometer at a wavelength of 280 nm, and the optical density was 0.1.
Antigen solution 1 was prepared as follows.

Leptospira hebdomadis hepdomadis strain was grown in Kortoff's medium in the same manner as autumn plague A strain. After washing, sonication treatment was performed. The obtained centrifuged supernatant was centrifuged at 280 nm.
Antigen solution 2 was prepared so that the optical density was 0.1 when measured at a wavelength of .

Mix 2 ml each of antigen solutions 1 and 2, and add 2 ml of the above-mentioned glutaraldehyde-treated microcapsules.
mixed with. After incubating for 1 hour at 37°C, 4
It was left standing in a refrigerator at ℃ for 18 hours.

Next, after washing twice with physiological saline containing 0.2% glycine +
0,15M+ containing 2ml of 3% bovine serum albumin
, monophosphate buffered saline (PBS pH=7.2)
Reagent A was obtained.

Performance test: The performance of this reagent A was preliminarily tested by the microtiter method using antisera of Autumnalis autumnalis A strain and Hepdomadis hebdomadinu strain prepared by the method described below.

The results obtained are shown in Table 1, in the "Reagent A" column.

From the preliminary tests shown in Table 1 and above, we were able to obtain the desired sensitivity (10240) for anti-hepdomadis and hepdomadis antibodies, but the sensitivity for anti-hepdomadis and desiccant A antibodies was the same as the desired sensitivity (10240). 10240) was not reached.

Therefore, a single-strain sensitization reagent having the desired sensitivity (10240) was prepared by sensitizing A. autumnalis A alone in advance. Add this to reagent A in volume ratio, reagent A
: Autumnalis sensitization reagent A single sensitization reagent = 3 = 1 were mixed to obtain reagent A'.

Similar to Reagent A, microtiter tests were performed using the two corresponding antisera. The obtained results are shown in Table 1.
It is shown in the "Reagent A'" column.

Reagent A (Autumnalis A and Hepdomadis
Reagent A' of the present invention obtained by mixing a single sensitizing reagent for A. autumnalis A.
In this case, the sensitivity to the anti-autumnalis/desire skin A antibody, which had not reached the target sensitivity in the preliminary test, increased to the target sensitivity. Therefore, the sensitivity level can be made the same for each antibody, and as a result, noise is reduced and antibody detection sensitivity is increased.

Example 2 Preparation of three bacterial strain sensitized microcapsule reagent B.

In the same manner as in Example 1, three strains, Rhesotosvira autumnalis strain A, Hebdomadis hebdomadis strain, and Icterohemoragies RGA strain, were grown in Kortov's medium. After washing, each sample was sonicated. Each centrifuged supernatant prepared to have an optical density of 0.1 at a wavelength of 280 nm was used as an antigen solution.

Antigen solution of three types of bacteria 2mA! (i7 mixed, mixed in microcapsules as in Example 1 and reacted, reagent Be
Obtained.

As in Example 1, a preliminary test was conducted by the microtiter method using antisera corresponding to three types of bacterial strains.

The results obtained are shown in Table 2, column "Reagent B".

As a result of the preliminary test in Table 2, we were able to obtain the desired sensitivity (10240) for anti-Autumnalis and Desi A antibodies, but we were able to obtain the desired sensitivity (10240) for anti-Icterohaemoragiae and RGA antibodies and anti-Hebdomadis and Hebdomadis antibodies. It was found that the sensitivity to the target did not reach the desired sensitivity (1024,0).

Therefore, a single-strain sensitization reagent having the desired sensitivity (10240)'1f was prepared in advance by sensitizing each of Icterohemolagiae RGA and Hebdomadis hebdomadis alone. This is used as reagent B, and the volume ratio is reagent B: Icterohemoragie RGA alone reagent: Hebdomadis hebdomadis strain alone reagent = 3:1:1
The mixture was mixed to obtain reagent B'.

Similar to Reagent B, a microtiter test was performed using three antisera.

The results obtained are shown in Table 2, in the column labeled "Reagent B/J."

Sense of purpose in preliminary test 111j (10240)K
The unreachable sensitivity for anti-Icterohemolagies RGA antibody and Hepdomadis hebdomadis antibody was improved to the desired sensitivity with reagent B' of the present invention. Therefore, the sensitivity level for each antibody was the same, and as a result, noise was reduced and three types of antibodies could be detected simultaneously with good reproducibility and high accuracy.

Reagents A (A') and B (B') prepared in Examples 1 and 2
) performance was evaluated by antibody titer using the following antiserum and the macro titer method.

(Preparation of antiserum) (a) Leptospirobacterium icterohemoragiae strain RGA (b) Leptospirobacterium icterohemolagiae strain A.

(c) Hephdomadis hebdomadis strain.

Rabbits were hyperimmunized with each of the strains (a), (b), and (e) above to prepare antisera.

Centrifuge the Cortos medium culture solution of each strain,
Float the precipitated bacterial cells in physiological saline.

This was subcutaneously injected into rabbits twice at intervals of 4 to 5 days.

Further intravenous injections were given nine times at intervals of 4 to 5 days. After 7 to 8 weeks had passed since the first subcutaneous injection and it was confirmed that the mice had a predetermined antibody titer, all blood was collected and antiserum for each strain was prepared.

(Test using microtiter method) Reagents A and A' that sensitized Rhesotospira bacterial cell components.

For B and B', antigen-antibody reactions were carried out using the microtiter method. Bright, female agglutination ff, and the observed anterior tube were determined to be positive, and the highest dilution of the antiserum against the three strains listed above was calculated and used as the antibody side.

For the antiserum of the three bacterial strains, dilute the test serum at 25 μl with 0.15 M IJ acid-buffered saline (PBS pH 7,2) at 2-fold intervals into each well of the microplate. A fold dilution series was created.

Next, 25 μl each of microcapsule reagents A, A', B, and B' sensitized with the Rhizotospira bacterial cell components were added.
The samples were collected using a Drono i4-, and sequentially dropped into the tube holes of the antiserum dilution series of the microplate. After shaking the microplate for 5 minutes to advance the antigen-antibody reaction, place it in the refrigerator at 4℃ for 1 hour.
It was left standing for 8 hours. Then take it out.

The agglutination image at the bottom of the tube was observed using a micrograte device on a light table, and the antibody titer was determined by the highest dilution ratio of the serum that showed agglutination.

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Claims (6)

[Claims] (1) Contains sensitized microcapsules after mixing two or more foreign antigens as the main microcapsules. In a group of microcapsules in which the sensitivity level to each antibody of the foreign antigen is uneven, at least one microcapsule sensitized solely with an antigen for which the sensitivity level is insufficient in the main microcapsules is mixed as an auxiliary microcapsule. Microcapsules for detecting multiple antibodies characterized by: (2) The microcapsule according to claim 1, wherein the foreign antigen is an antigen component of a different serotype. (3) The microcapsule according to claim 2, wherein the antigen component is Leptospira bacteria. (4) main microcapsules sensitized after mixing two or more foreign antigens exhibiting non-uniform sensitivity levels to antibodies;
A multi-antibody testing method characterized in that an antigen-antibody reaction is carried out using a mixed microcapsule obtained by mixing at least one auxiliary microcapsule sensitized with an antigen with insufficient sensitivity level. (5) The test method according to claim 4, wherein the foreign antigen is an antigen component of a different serotype. (6) The test method according to claim 5, wherein the antigen component is Leptospira bacteria.